A mass spectrometer is a type of instrument that determines the mass-to-charge ratios of the ionized constituents of a sample. There are several different types of mass spectrometers. In a time-of-flight mass spectrometer (TOF MS), the sample to be analyzed is first ionized and then exposed to a voltage pulse that accelerates the ions through a vacuum along a path toward a detector. The lower the mass-to-charge ratio of the ion, the more it will accelerate and, therefore, the earlier it will arrive at the detector. Thus, a TOF MS segregates the ions liberated from the sample by mass-to-charge ratio based on their arrival time at the detector.
The detector converts the impacts of the ions on the detector into electrons. One or more ions may hit the detector at any given time. There is a statistical correlation between the number of ions hitting the detector and the number of electrons generated. The number of electrons leaving the detector in a given time interval is converted to a voltage that is digitized by an analog-to-digital converter (ADC).
The greater the mass-to-charge ratio of the ion, the longer the flight time to the detector. The relationship between the flight time and the mass-to-charge ratio can be written in the form:time=k√{square root over ((m/z))}+c where k is a constant related to flight path and ion energy, m is mass, z is charge, and c is a small delay time that may be introduced by the acceleration and/or detection electronics.
Thus, on average, the signal output from the ADC at any given instant is proportional to the number of ions reaching the detector at that instant. The delay for an ion to reach the detector (i.e., the time of flight to reach the detector after the acceleration pulse) is proportional to the square root of the mass-to-charge ratio of the ion. Hence, the output of the ADC can be processed to generate a plot (or spectrum) of the concentration of ions from the sample as a function of mass-to-charge ratio (hereinafter m/z). Specifically, the time of flight statistically correlates to the m/z of the ion and the number of ions hitting the detector at that instant correlates statistically to the relative concentration of ions ionized from the sample of that particular m/z. For purposes of digitally processing the detector data to generate a spectrum, the range of delay times is divided into discrete “bins” and the output of the ADC in each time bin is analyzed to generate a data point. The collection of these data points is used to generate the mass spectrum.
The mass resolution of the spectrometer depends in part on the time between the bins into which the flight time measurements are divided. The resolution as to concentration of the ions of a given m/z (i.e., dynamic range) depends in part on the resolution of the ADC output (i.e., the number of bits of output of the ADC).
Hence, the time of a peak in the spectrum corresponds to the m/z of the ions and the amplitude of that peak corresponds to the abundance (or concentration or number) of ions having that m/z.
Generally, the identity of the constituents of a sample can be accurately determined from TOF MS, especially with advance knowledge of what should be expected. Therefore, TOF MS can be used to determine the molecular constituents of a sample and the abundance or concentration of those constituents. However, it is conceivable that two different ions in a sample could have the same mass-to-charge ratio or at least that the difference between their mass-to-charge ratios is below the resolution of the system so that they are indistinguishable from each other by the TOF MS.
Typically, the amount of ions liberated by a single acceleration pulse and measured by the detector as discussed above (commonly referred to as a transient or transient response) is too small to provide a statistically accurate mass spectrum of the sample. Hence, the transient measurement is repeated a number of times (usually on the order of hundreds to tens of thousands) and the data from the multiple transients is combined (e.g., summed) to generate statistically relevant amounts of data at an acceptable signal-to-noise (S/N) ratio. The plurality of transients measured to generate a reported mass spectrum will sometimes hereinafter be referred to as a scan.
Generally, the lower the concentration of an analyte of interest, the greater the number of TOF MS sums (i.e., transients) needed to achieve a desired S/N ratio. Thus, the number of transients summed per spectrum (the number of transients per scan) usually is set as a function of the lowest expected abundance or concentration of an analyte of interest.
The time that must be permitted for each transient is a function of the highest m/z ion that might exist in the sample. Quite simply, the highest m/z ion in the sample will arrive at the latest time. The time provided for each transient measurement (i.e., the time between the voltage pulses that accelerate the ions) must be at least as long as it would take for the slowest-traveling ion to arrive at the detector.
A typical maximum allowed time of flight for a transient might be on the order of 100 microseconds or so. Thus, if we assume (1) a typical number of transients to obtain statistically relevant data, such as 10,000 transients, (2) a typical number of data points (i.e., time bins) per transient, such as 100,000, and (3) four bytes per data point to represent the number of ion impacts detected in each time bin, that results in a data rate of 4 gigabytes per second.
Transferring data to a processor at this rate is not possible at reasonable expense with current computer technology. Furthermore, the amount of memory capacity that would be necessary in a TOF MS to store this data for later, off-line processing also is not commercially practical. Therefore, rather than storing or processing the spectra resulting from each transient individually, the data from all of the transients is summed and only the sum is stored. When all of the transients have been processed, the sum is used to generate a single, consolidated mass spectrum.
Typically, in TOF MS, the output spectrum is a plot of concentration (or abundance or number of ions) on the vertical axis as function of time (which is correlated to √{square root over (m/z)}) on the horizontal axis. A typical spectrum consists of a plurality of populations of ions of a given m/z, often referred to as mass peaks.
In addition, a TOF MS system often is a part of a larger system that couples the TOF MS instrument with another instrument that also time-segregates the sample to provide a second dimension of data in the ultimate output of the system. For example, the sample introduced at the input end of a TOF MS might be the output of a gas or liquid chromatograph, a quadrupole mass filter, a collision cell, a MALDI (Matrix Assisted Laser Desorption Ionization) stage, or an ion trap mass spectrometer.
A gas or liquid chromatograph, for instance, may be placed before the TOF MS so as to provide a sample that has already been chemically separated as a function of time of arrival at the output of the chromatograph (i.e., at input to the TOF MS). The output of a chromatograph commonly might have peaks of analytes arriving at its output that are seconds to a few minutes wide separated by many minutes of background noise.
As another example, a quadrupole mass filter is an adjustable mass filter that can be set to allow ions within a particular m/z range to pass through. The time required for such filters to transition between m/z ranges could be on the order of microseconds to milliseconds. A collision cell may be further included between the quadrupole mass filter and the TOF MS analyzer. The optimal dwell time for integration of the TOF MS signal for a particular quadrupole filter m/z range setpoint is a function of the incoming ion signal intensity in that m/z range. Generally, this will be different for different m/z ranges and also typically will be time-variant.
In an exemplary MALDI stage, a sample of a chemical compound that is sensitive to laser light (the matrix) is hit with a pulse of laser light to superheat the sample to cause a portion of the sample to be desorbed and become ions. The optimal values for the number of times the sample is hit with the laser pulse, the duration of the pulse, the power of the pulse, how often the laser is moved to strike a new portion of the sample, and how long to integrate the data in the MALDI can depend on many factors. In turn, the duration of signals associated with ionized sample components in the output of the MALDI stage and the intervals between those signals can vary significantly.
In ion trap mass spectrometry, ions are captured in a storage device (trap) and mass-selectively ejected from the trap.
In all of the aforementioned potential preceding stages to a TOF MS, the output (which, of course, is the input to the TOF MS) is already separated by either chemical properties or mass as a function of time. Therefore, such combined systems can provide greater mass resolution, greater mass accuracy, greater component resolution, and greater concentration accuracy and/or resolution.
In such combined systems, the TOF MS stage generates a plurality of consecutive, time-separated mass spectra of the time-varying input sample in order to extract the most information from the sample.